2.7 Flashcards
DNA replication is a…
semi-conservative process
when a new double-stranded DNA molecule is formed:
- One strand will be from the original template molecule
- One strand will be newly synthesised
DNA replication occurs because
each nitrogenous base can only pair with its complementary partner (CG) (AT)
Consequently, when DNA is replicated by the combined action of helicase and DNA polymerase:
- Each new strand formed will be identical to the original strand separated from the template
- The two semi-conservative molecules formed will have an identical base sequence to the original molecule
The theory that DNA replication was semi-conservative was confirmed by which experiment?
Meselson-Stahl experiment in 1958
Prior to this experiment, three hypotheses had been proposed for the method of replication of DNA:
- Conservative Model
- Semi-Conservative Model
- Dispersive Model
Conservative Model
An entirely new molecule is synthesised from a DNA template (which remains unaltered)
Semi-Conservative Model
Each new molecule consists of one newly synthesised strand and one template strand
Dispersive Model
New molecules are made of segments of new and old DNA
Meselson and Stahl were able to experimentally test the validity of these three models using…
radioactive isotopes of nitrogen
Meselson and Stahl procedure with nitrogen
DNA molecules were prepared using the heavier 15N and then induced to replicate in the presence of the lighter 14N and DNA samples were then separated via centrifugation to determine the composition of DNA in the replicated molecules
The results after two divisions supported the semi-conservative model of DNA replication
- After one division, DNA molecules were found to contain a mix of 15N and 14N, disproving the conservative model
- After two divisions, some molecules of DNA were found to consist solely of 14N, disproving the dispersive model
The process of DNA replication is coordinated by two key enzymes
helicase and DNA polymerase
Helicase
- Helicase unwinds the double helix and separates the two polynucleotide strands
- It does this by breaking the hydrogen bonds that exist between complementary base pairs
- The two separated polynucleotide strands will act as templates for the synthesis of new complementary strands
DNA Polymerase
- DNA polymerase synthesises new strands from the two parental template strands
- Free deoxynucleoside triphosphates (nucleotides with 3 phosphate groups) align opposite their complementary base partner
- DNA polymerase cleaves the two excess phosphates and uses the energy released to link the nucleotide to the new strand
The polymerase chain reaction (PCR)
is an artificial method of replicating DNA under laboratory conditions
PCR is used to
amplify large quantities of a specific sequence of DNA from an initial minute sample. Each reaction doubles the amount of DNA – a standard PCR sequence of 30 cycles creates over 1 billion copies (230)
PCR occurs in a thermal cycler and uses variations in temperature to control the replication process via three steps:
- Denaturation – DNA sample is heated (~90ºC) to separate the two strands
- Annealing – Sample is cooled (~55ºC) to allow primers to anneal (primers designate sequence to be copied)
- Elongation – Sample is heated to the optimal temperature for a heat-tolerant polymerase (Taq) to function (~75ºC)
Taq polymerase is an enzyme isolated from
the thermophilic bacterium Thermus aquaticus
Thermus aquaticus optimal temperature
As this enzyme’s optimal temperature is ~75ºC, it is able to function at the high temperatures used in PCR without denaturing
Transcription
the process by which an RNA sequence is produced from a DNA template
transcription: role of DNA polymerase
RNA polymerase separates the DNA strands and synthesises a complementary RNA copy from one of the DNA strands